Joyce N. Malinga, JanKreuze, Maria Soto, March Ghislain, Simon - - PowerPoint PPT Presentation

EVALUATION OF TRANSGENIC SWEETPOTATO (Ipomoea batatas L.) LINES AGAINST SWEETPOTATO VIRUS DISEASE(SPVD) COMPLEX

• Joyce N. Maling’a, JanKreuze, Maria Soto, March

Ghislain, Simon Gichuki, Michela Akhwale, Laura Karanja, Alice Dok and Grace Olweny

• KALRO
• Donald Danforth Plant Science Center
• International Potato Centre

Background

Importance of sweetpotato (What message)

• Sweetpotato is a main staple crop for millions
• f subsistence farmers in Africa. It’s seventh

ranked most important food crop world wide (FAO 2012)

• Its grown for its storage roots for food

security and income generation (Ngailo et al, 2013)

Production trends

• Diseases (viruses) & Pests
• Severe shortage of healthy planting

material

• Low yielding varieties
• Poor agronomic practices

Constraints to sweetpotato production & utilization in Kenya

• Sweet potato Virus Disease complex (SPVD) is

a synergistic dual virus infection caused by Sweet Potato Chlorotic Stunt Virus(SPCSV) and Sweet Potato Feathery Mottle virus[SPFMV] (Gibson, et al 1998, Mukasa, et al 2006).

• Hard to control,
• Spread of SPVD is enhanced by poor farming

practices

Justification

Comparison of healthy and SPVD infected plant

Sweetpotato Virus complex

Statement of Problem

• SPVD leads to-50-98% crop loss (Ngailo, 2013)
• Use of local sweetpotato varieties that are low

yielding and susceptible to viruses

• Major challenges encountered in conventional

breeding

Justification

• Difficulty in controlling Sweetpotato viral diseases
• Genetic engineering using gene silencing is a promising
• pportunity
• Incorporation of resistant genes into susceptible but

preferred Sweetpotato varieties or landraces-strategy for long term disease control (Fraile et al 2011)

The strategy

• siRNAI
• The strategy of inducing virus resistance through

RNAi using transgene constructs designed to produce double stranded RNA corresponding to the corresponding virus(es) has been effectively used to control plant viral pathogens in cassava and other crops and offers an attractive option of integrating virus resistance into farmer-preferred cultivars.

Specific Objectives

I. To evaluate agronomic traits of transgenic and non-transgenic (wild type) Sweetpotato lines in Confined field trial (CFT).

• II. To evaluate the virus expression of SPVD of

transgenic and non-transgenic (wild type) Sweetpotato lines in the CFT

• III. To assess transgenic sweetpotato lines for the

presence of SPVD resistant trans-genes through molecular screening.

• IV. To identify the SPCSV and SPFMV strains and

determine concentration of virus titres affecting the events using RT-PCR

Methodology

Plant Materials (When message communicated)

• Plant materials consisted of 17 gene events(lines)

made from gene constructs PC 227 and PC224 from PI112253 at Danforth Plant science centre, USA and 3 gene events from Huachano, wild type at International Potato Centre (CIP), Helsinki.

• Ejumula - a susceptible variety and Naspot 1(tolerant

Variety) were used in the study.

• A total of 1440 plantlets grouped into 24 Sweetpotato

lines were used in the experimental set up.

Plant Materials

• The Sweetpotato in-vitro plantlets were

transported to Kenya Agricultural and Livestock Organization, (KALRO) Kakamega screen house under bio-safety conditions under the supervision of a KEPHIS inspector.

• The in-vitro plantlets were washed to remove

from culture media, then acclimatized and hardened in the screen house for eight weeks before transferring to the CFT

Experimental site: Confined Field Trial – (when)

The plantlets were transported from screen house into Confined Field Trial located at KALRO, Kakamega Centre for experimentation. Susceptible Sweetpotato varieties namely Ejumula and Kenspot 4 were used as the spreader varieties due to their high susceptibility to virus infection while Naspot1 was used as positive control.

Transgenic Virus-Resistant Sweetpotato Confined Field Trial Plot Sketch KALRO NRI Kakamega

Transgenic and Control sweetpotato Plants SPVD Infector sweetpotato Plants Guard Row sweetpotato Plants Ejumula)

Incineration Pit Gate Guard House Stor e Chain linked fence Foot Bath

Experimental Layout

Data Collection

• 1. Comparative Morpho-agronomic characterisation of transgenic

events versus Wild Type (WT)

• 2. Incidence of Virus Infected plants
• 3. Comparative virus scores of transgenic events VS WT

Monthly virus scores were done on each plot using the scale of 1 – 9 where:

1 -no virus infestation, 2 - unclear virus symptoms 3- clear virus symptoms 5% of plants affected 4-clear virus symptoms 6-15% of planted affected 5-clear virus symptoms 16-33% of plants affected 6-clear virus symptoms 34-66% of plants affected 7-clear virus symptoms 67-99% of plants affected 8- clear virus symptoms all plants affected 9--clear virus symptoms, all plants affected and stunted, almost dead plants.[Here you need to describe in more detail what exactly each score corresponds to. 1 = no symptoms, 2 = etc.]

Evaluating SPVD resistance cont.

• The incidence and severity of SPVD was

evaluated every two weeks for 200 weeks after planting.

• Severity was scored on a scale of 1-5 as

described by Hahn (1979).

• SPVD incidence was expressed as

percent of diseased plants compared to the total number of plants present in the plot (Guiterrez et al. 2003).

RESULTS

NASPOT 1 - Resistant check

Huachano-WT

pCIP41

pCIP41

pCIP41

pC127

pC127

pC127

pC127

pC127

Table 1.1 Comparative agronomic performance and SPVD reactions recorded for transgenic lines and PI531122 WT at kakamega harvested after 5 months at Kakamega and over 3 replicates in October 2016

Entr y Transgenic line using C127 construct Si RNA levels Plants at harvest Root yield t ha-1 No of Mktable roots No Unmkt roots Wt Mkt kg/plot Wt Unmkt Kg/plot Above ground biomass kg/plot Incidence 0f SPVD (%) Overall plot Sevirity scores(1- 9)(1- R;9VS) 1 2 3 4 >5 10 C127-Y5 + 20 23 38.7 50.3 37.7 8.3 64 100 4.5 60 37.5 1 C127-7 +++ 20 17.7 33.7 102 20.3 15 70.7 100 6 35 45 7.5 12.5 5 C127-23 +++ 20 11.3 20 52.3 13 9.7 79.3 100 6 25 67.5 0 12 C127-Y11 +++ 19.3 5.5 8.3 41 5 5.7 80.3 97.5 4 25 75 3 C127-16 ++++ 19.3 7.7 11 69.3 8 7 52.3 95 7 30 30 35 4 C127-18 ++++ 20 10.8 13 72.3 10.7 11 65.7 100 7 2.5 30 50 17.5 6 C127-27 ++++ 17.3 4 0.7 25.7 1.3 5.3 46.3 80 8 15 65 8 C127-74 ++++ 20 12.5 16 102.7 12.3 0.7 48 100 6 7.5 57.5 35 14 C127-Y20 ++++ 19.7 7.6 11 70.7 6.3 8.7 100.6 100 4 80 12.5 7.5 16 C127-Y40 ++++ 20 11.5 28.3 35 17.3 5.7 81.7 100 6 10 47.5 27.5 15 2 C127-14 +++++ 20 9.7 12 63.3 11.7 7.7 86.3 100 4.5 75 10 15 7 C127-40 +++++ 20 12.7 17.7 81 14.3 11 84.3 100 4.5 75 22.5 9 C127-83 +++++ 18.7 4.2 3.7 32.7 2.7 5 50.7 90 4 55 27.5 2.5 2.5 11 C127-Y10 +++++ 19.7 15.3 27.7 51.3 22 8.3 83.3 97.5 6 97.5 13 C127-Y13 +++++ 20 14.2 23.7 62 20.3 8 72.3 100 4 47.5 37.5 15 15 C127-Y21 +++++ 20 9.7 13.3 74.7 10.3 9 101.7 100 3 85 12.5 17 C127-P4 +++++ 20 17.2 27 156.3 19.3 15 104.3 100 4 65 15 20 21 PI 531122 None 19.3 10.8 20.3 41.3 13.3 7.3 43 95 8 1.5 6.5 11 Mean 19.63 11.41 18.12 65.77 13.66 8.24 73 97.5 5.4 6.9 5.9 3.6 2.3

LsD=P<0.0 5) 1.7 9.3 16.4 60.1 85

8.5

1.2

Entr y Transgenic line using C1P41 construct Si RNA levels Plants at harvest Root yield t ha-1 Mktable roots Unmkt roots Wt Mkt kg/plo t Wt Unmkt Kg/plo t Above ground biomass kg/plot Incide nce 0f SPVD Percent plants suffering severity scores (1-9; 1- resistant, 9 severe) Overall plot 1 2 3 4 >5 18 CIP41-1 ++ 18.7 2.1 2.7 18.3 1 3 50.3 18 8 0.5 10 77.5 19 CIP41-9 + 19.3 0.9 31.7 1.7 37 19 9 10. 85 20 CIP41-23 + 19 2.2 8.7 13.3 2.3 1.7 71.7 19 8 10 85 22 Huachano None 19.7 3.9 5 26 3 4.7 74 19.5 8 15 85 Mean 19.2 2.3 4.1 22.3 1.6 2.8 58.3 18.9 8.3

• - 0.1

3.0 83.1 Std dev 0.4 1.2 3.7 8.1 1.3 1.4 17.7 0.6 0.5 0.0 0.0 0.3 0.0 0.8

Table 1.2 Comparative agronomic performance and SPVD reactions recorded for Transgenic lines and Huachano WT at Kakamega harvested after 5 months at Kakamega and over 3 replicates in December 2016

Entr y Transgeni c line using C127 construct Si RNA levels Plants at harvest Root yield t ha-1 No of Mktable roots No Unmkt roots Wt Mkt kg/plot Wt Unmkt Kg/plot Above ground biomass kg/plot Incidenc e 0f SPVD (%) Overa ll plot Sevirit y score s(1- 9)(1- R;9VS ) 1 2 3 4 >5 8 C127-74 ++++ 20 12.5 16 102.7 12.3 0.7 48 100 6 7.5 57.5 35 14 C127-Y20 ++++ 19.7 7.6 11 70.7 6.3 8.7 100.6 100 4 80 12.5 7.5 16 C127-Y40 ++++ 20 11.5 28.3 35 17.3 5.7 81.7 100 6 10 47.5 27.5 15 2 C127-14 +++++ 20 9.7 12 63.3 11.7 7.7 86.3 100 4.5 75 10 15 15 C127-Y21 +++++ 20 9.7 13.3 74.7 10.3 9 101.7 100 3 85 12.5 0 17 C127-P4 +++++ 20 17.2 27 156.3 19.3 15 104.3 100 4 65 15 20 21 PI 531122 None 19.3 10.8 20.3 41.3 13.3 7.3 43 95 8 7.5 32.5 55

Table 1.3 Comparative agronomic performance and SPVD reactions recorded for the best 6 transgenic lines, the untransformed parent genotype and NASPOT 1 at kakamega harvested after 5 months at Kakamega and over 3 replicates in December 2016

CONCLUSIONS

 Partial control of SPVD has been demonstrated

The constructs were designed to control SPCSV and SPFMV Greater analysis of the samples required to determine what viruses and what titre is present in the plants Possible that one or more of the target viruses have been effectively suppressed – needs to be ascertained When known what viruses are present in samples – then new improved constructs can be designed

Acknowldeg ement DDPSC CIP KALRO NBA